Multifunctional primers for paired sequencing reads

Abstract

The invention provides methods of generating forward and reverse reads of an immobilized single-stranded target nucleic acid using multifunctional primers. A multifunctional primer serves to initiate separate syntheses of first and second complementary strands to the single-stranded target nucleic acid, and to tether the first complementary strand to the immobilized target nucleic acid. The first complementary strand serves to provide a primer binding site and template for an extension product to generate a reverse sequencing read. The second complementary strand serves to displace the first complementary strand from duplexing with the target nucleic acid. The first or second complementary strand can provide a forward sequence read.

1 . A method of obtaining paired sequencing reads of a target nucleic acid, comprising
 (a) contacting a single-stranded target nucleic acid flanked at its 5′ end by a 5′ adapter immobilized to a support and at its 3′ end by a 3′ adapter with a forward primer comprising a 3′ target binding region and a 5′ stem-loop to form a reaction mix, wherein the target binding region binds the 3′ adapter;   (b) ligating a free 5′ end of the stem loop to the 3′ adapter;   (c) conducting an extension reaction, wherein the target binding region primes synthesis of a first complementary strand to the target nucleic acid and optionally provides a sequencing read of the target nucleic acid;   (d) contacting the reaction mix with a loop primer that anneals to the loop region of the stem loop;   (e) conducting an extension reaction, wherein the loop primer primes synthesis of a second complementary strand to the target nucleic acid, and the first complementary strand is displaced from duplexing with the target nucleic acid;   (f) contacting the reaction mix with a reverse primer, which anneals to a complement of the 5′ adapter at the 3′ end of the displaced first complementary strand; and   (g) conducting a sequencing-by-extension reaction, wherein the reverse primer primes synthesis of a strand complementary to the displaced first complementary strand and provides a sequencing read of the displaced first complementary strand.   
     
     
         2 . The method of  claim 1 , further comprising before step (a) contacting the single-stranded target nucleic acid with a second forward primer comprising a target binding region, which binds to a complementary site in the 3′ adapter;
 conducting a sequencing-by-extension reaction wherein the target binding region of the second forward primer primes synthesis of a further complementary strand of the target nucleic acid and provides a sequencing read of the target nucleic acid; and 
 displacing the further complementary strand from duplexing with the target nucleic acid. 
 
     
     
         3 . The method of  claim 1 , wherein step (c) is a sequencing-by-extension reaction that provides a sequence read of the target nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein step (c) is performed before step (b). 
     
     
         5 . A method of obtaining paired sequencing reads of a target nucleic acid, comprising
 (a) contacting a single-stranded target nucleic acid flanked at its 5′ end by a 5′ adapter immobilized to a support and at its 3′ end by a 3′ adapter with a forward primer comprising a 3′ target binding region and a 5′ stem loop to form a reaction mix, wherein the target binding region binds the 3′ adapter and wherein the target binding region comprises a cleavable site;   (b) ligating a free 5′ end of the stem loop to the 3′ adapter;   (c) conducting a sequencing-by-extension reaction, wherein the target binding region primes synthesis of a first complementary strand to the target nucleic acid and provides a sequencing read of the target nucleic acid; cleaving the cleavable site, and conducting a further extension reaction wherein the remainder of the target binding region after cleavage primes synthesis of a further complementary strand to the target nucleic acid;   (d) contacting the reaction mix with a loop primer that anneals to the loop region of the stem loop;   (e) conducting an extension reaction wherein the loop primer primes synthesis of a second complementary strand to the target nucleic acid, which displaces the further complementary strand from duplexing with the target nucleic acid;   (f) contacting the reaction mix with a reverse primer, which anneals to a complement of the 5′ adapter at the 3′ end of the displaced further complementary strand; and   (g) conducting a sequencing-by-extension reaction, wherein the reverse primer primes synthesis of a strand complementary to the displaced further complementary strand and provides a sequencing read of the displaced further complementary strand.   
     
     
         6 .- 7 . (canceled) 
     
     
         8 . A method of obtaining paired sequencing reads from a target nucleic acid, comprising
 (a) contacting a single-stranded target nucleic acid flanked at its 5′ end by a 5′ adapter immobilized to a support and at its 3′ end by a 3′ adapter with a forward primer comprising first and second 3′ target binding regions flanking an intervening region to form a reaction mix, wherein the first and second target binding regions bind complementary sites in the 3′ adapter molecule, the respective sites being distal and proximal to the 3′ end of the 3′ adapter;   (b) conducting an extension reaction, wherein the first target binding region primes synthesis of a first complementary strand to the target nucleic acid and optionally provides a sequencing read of the target nucleic acid;   (c) conducting a second extension reaction wherein the second target binding region primes synthesis of a second complementary strand to the target nucleic acid, which displaces the complementary strand from duplexing with the target nucleic acid;   (d) contacting the reaction mix with a reverse primer, which anneals with a complement of the 5′ adapter at the 3′ end of the displaced first complementary strand; and   (e) conducting a sequencing-by-extension reaction, wherein the reverse primer primes synthesis of a strand complementary to the displaced first complementary strand and provides a sequencing read of the displaced first complementary strand.   
     
     
         9 . The method of  claim 8 , further comprising before step (a) contacting the single-stranded target nucleic acid with a second forward primer comprising a target binding region, which binds to a complementary site in the adapter molecule at the 3′ end of the target nucleic acid;
 conducting a sequencing-by-extension reaction wherein the target binding region of the second forward primer primes synthesis of a further complementary strand of the target nucleic acid and provides a sequencing read of the target nucleic acid; and 
 displacing the further complementary strand from duplexing with the target nucleic acid. 
 
     
     
         10 . The method of  claim 8 , wherein the intervening region comprises an inverted nucleotide or a non-nucleotide linkage. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 8 , wherein the second target binding region is extension blocked and the method further comprises removing the block to extension before performing the extension primed by the second target binding region. 
     
     
         13 . The method of  claim 12 , wherein the second target binding region is extension blocked by a 3′ terminal phosphate group and removing the block comprising treating with a phosphatase to remove the phosphate. 
     
     
         14 . The method of  claim 8 , wherein the second extension is performed with a strand-displacing polymerase. 
     
     
         15 . The method of  claim 8 , wherein the 3′ adapter further comprises a sample index between the complementary sites for the first and second target binding regions. 
     
     
         16 . The method of  claim 8 , wherein the 5′ or 3′ adapter or both further comprises a molecular index and/or a sample index. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 16 , wherein the single-stranded nucleic acid flanked by the 5′ and 3′ adapters is produced by emulsion amplification. 
     
     
         19 . The method of  claim 18 , further comprising breaking the emulsion before performing step (a). 
     
     
         20 . The method of  claim 18 , wherein the support to which the single-stranded target nucleic acid is immobilized was used in emulsion PCR to generate the single-stranded target nucleic acid flanked by 5′ and 3′ adapters, or was attached after emulsion PCR. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein the single-stranded target nucleic acid immobilized to a support is one of a clonal population of such target nucleic acids immobilized to the same support. 
     
     
         23 . The method of  claim 22 , wherein the support is a bead, and/or and addressable region within an array. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 23 , wherein the sequencing is by ion torrent, ultima sequencing, and/or single molecule sequencing, or clonal sequencing. 
     
     
         26 .- 27 . (canceled) 
     
     
         28 . The method of  claim 8 , wherein the length of the target nucleic acid exceeds the maximum read length of the sequencing method and/or wherein the target nucleic acid is about 170 nucleotides with the maximum read length of the sequencing method about 150 nucleotides of the target nucleic acid. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 28 , wherein the sequencing reads have a non-overlapping region. 
     
     
         31 . The method of  claim 30 , further comprising determining a composite sequencing read from the forward and reverse sequencing reads in which any positions of discordance between forward and reverse sequencing reads are left open or as alternative nucleotides occupying the position in the forward and reverse sequencing reads. 
     
     
         32 .- 36 . (canceled)